The objective of this project is to elucidate the mechanism by which endogenous opiate peptides are metabolized. This research will focus on elucidating the physiological role of two enkephalin degrading enzymes; a membrane endopeptidase (enkephalinase) cleaving enkephalins at the gly-phe bond and a recently described membrane bound puromycin insensitive aminopeptidase. Multiple forms of the membrane endopeptidase have been demonstrated to exist in rat brain. These multiple forms will be isolated and compared to each other in terms of their physical properties. These studies are designed to elucidate differences in these multiple enkephalinase forms due to differences in either primary structure or glycosylation. The substrate specificity of each enkephalinase form will be examined in terms of various opiate peptides and other biologically active non-opiate peptides. Monoclonal antibodies specific for each of the enkephalinase forms will be produced and used in immunohistochemical localization studies. These studies are designed to determine whether the multiple enkephalinase forms have a differential distribution in brain and to correlate enkephalinase distribution with that of opiate peptides and opiate receptors. Studies with the membrane puromycin insensitive aminopeptidase will parallel those to be conducted with the endopeptidase. These studies will include characterization of the enzyme in terms of its physical properties and substrate specificity, and the use of antibodies produced against the enzyme for immunohistochemical localization studies. Taken together these studies should help to elucidate the physiological role of these peptidases in the metabolism of endogenous enkephalins and other opiate peptides. In addition, these studies should help in assessing the importance of these enzymes in regulating opiate peptide levels "in vivo".